Peptides effective in the treatment of tumors and other conditions requiring the removal or destruction of cells

ABSTRACT

The embodiments include methods of treating conditions requiring removal or destruction of cellular elements, such as benign or malignant tumors in humans, using compounds based on small peptides. The method includes, but is not limited to, administering the compounds intramuscularly, orally, intravenously, intrathecally, intratumorally, intranasally, topically, transdermally, etc., either alone or conjugated to a carrier.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.11/680,119, filed on Feb. 28, 2007, now U.S. Pat. No. 8,067,378, whichclaims benefit of U.S. Provisional Application No. 60/776,933, filedFeb. 28, 2006, the disclosures of both of which are hereby incorporatedby reference in their entireties.

BACKGROUND

1. Field of the Embodiments

The embodiments include methods of treating conditions requiring removalor destruction of cellular elements, such as benign or malignant tumorsin humans, using compounds based on small peptides. The method includes,but is not limited to, administering the compounds intramuscularly,orally, intravenously, intrathecally, intratumorally, intranasally,topically, transdermally, etc., either alone or conjugated to a carrier.

2. Description of Related Art

The essence of many medical treatments and procedures involves theremoval or destruction of harmful or unwanted tissue. Examples of suchimportant treatments include the surgical removal of cancerous growths,the destruction of metatastic tumors through chemotherapy, and thereduction of glandular (e.g. prostate) hyperplasia. Other examplesinclude the removal of unwanted facial hair, the removal of warts, andthe removal of unwanted fatty tissue.

There is an obvious need for an effective agent that will destroy andhence either facilitate the removal of or inhibit the further growth ofharmful or unwanted cells and tissue but will have mainly local effectsand minimal or absent systemic toxicity.

Classes of such agents are disclosed in pending U.S. patent applicationSer. No. 10/092,934, entitled: Methods of Treating Tumors and RelatedConditions Using Neural Thread Proteins, Ser. No. 10/153,334, entitled:Peptides Effective In The Treatment Of Tumors And Other ConditionsRequiring The Removal Or Destruction Of Cells; Ser. No. 10/198,069,entitled: Peptides Effective In The Treatment Of Tumors And OtherConditions Requiring The Removal Or Destruction Of Cells; Ser. No.10/198,070, entitled: Peptides Effective In The Treatment Of Tumors AndOther Conditions Requiring The Removal Or Destruction Of Cells, Ser. No.10/294,891 entitled: Peptides Effective In The Treatment Of Tumors AndOther Conditions Requiring The Removal Or Destruction Of Cells; and Ser.No. 10/920,313 entitled: Peptides Effective In The Treatment Of TumorsAnd Other Conditions Requiring The Removal Or Destruction Of Cells, thedisclosures of each of which are incorporated by reference herein intheir entirety.

Disclosed herein are composites, fragments and subsequences of one suchpeptide agent (SEQ ID NO. 1:Ile-Asp-Gln-Gln-Val-Leu-Ser-Arg-Ile-Lys-Leu-Glu-Ile-Lys-Arg-Cys-Leu)that also are useful in treating tumors and other conditions requiringremoval or destruction of cells.

Cancer is an abnormality in a cell's internal regulatory mechanisms thatresults in uncontrolled growth and reproduction of the cell. Normalcells make up tissues, and when these cells lose their ability to behaveas a specified, controlled, and coordinated unit, (dedifferentiation),the defect leads to disarray amongst the cell population. When thisoccurs, a tumor is formed.

Benign overgrowths of tissue are abnormalities in which it is desirableto remove cells from an organism. Benign tumors are cellularproliferations that do not metastasize throughout the body but do,however, cause disease symptoms. Such tumors can be lethal if they arelocated in inaccessible areas in organs such as the brain. There arebenign tumors of organs including lung, brain, skin, pituitary, thyroid,adrenal cortex and medulla, ovary, uterus, testis, connective tissue,muscle, intestines, ear, nose, throat, tonsils, mouth, liver, gallbladder, pancreas, prostate, heart, and other organs.

Surgery often is the first step in the treatment of cancer. Theobjective of surgery varies. Sometimes it is used to remove as much ofthe evident tumor as possible, or at least to “debulk” it (remove themajor bulk(s) of tumor so that there is less that needs to be treated byother means). Depending on the cancer type and location, surgery mayalso provide some symptomatic relief to the patient. For instance, if asurgeon can remove a large portion of an expanding brain tumor, thepressure inside the skull will decrease, leading to improvement in thepatient's symptoms.

Not all tumors are amenable to surgery. Some may be located in parts ofthe body that make them impossible to completely remove. Examples ofthese would be tumors in the brainstem (a part of the brain thatcontrols breathing) or a tumor which has grown in and around a majorblood vessel. In these cases, the role of surgery is limited due to thehigh risk associated with tumor removal.

In some cases, surgery is not used to debulk tumor because it is simplynot necessary. An example is Hodgkin's lymphoma, a cancer of the lymphnodes that responds very well to combinations of chemotherapy andradiation therapy. In Hodgkin's lymphoma, surgery is rarely needed toachieve cure, but almost always used to establish a diagnosis.

Chemotherapy is another common form of cancer treatment. Essentially, itinvolves the use of medications (usually given by mouth or injection)which specifically attack rapidly dividing cells (such as those found ina tumor) throughout the body. This makes chemotherapy useful in treatingcancers that have already metastasized, as well as tumors that have ahigh chance of spreading through the blood and lymphatic systems but arenot evident beyond the primary tumor. Chemotherapy may also be used toenhance the response of localized tumors to surgery and radiationtherapy. This is the case, for example, for some cancers of the head andneck.

Unfortunately, other cells in the human body that also normally dividerapidly (such as the lining of the stomach and hair) also are affectedby chemotherapy. For this reason, many chemotherapy agents induceundesirable side effects such as nausea, vomiting, anemia, hair loss orother symptoms. These side effects are temporary, and there existmedications that can help alleviate many of these side effects. As ourknowledge has continued to grow, researchers have devised newerchemotherapeutic agents that are not only better at killing cancercells, but that also have fewer side effects for the patient.

Chemotherapy is administered to patients in a variety of ways. Someinclude pills and others are administered by an intravenous or otherinjection. For injectable chemotherapy, a patient goes to the doctor'soffice or hospital for treatment. Other chemotherapeutic agents requirecontinuous infusion into the bloodstream, 24 hours a day. For thesetypes of chemotherapy, a minor surgical procedure is performed toimplant a small pump worn by the patient. The pump then slowlyadministers the medication. In many cases, a permanent port is placed ina patient's vein to eliminate the requirement of repeated needle sticks.

Radiation therapy is another commonly used weapon in the fight againstcancer. Radiation kills cancer by damaging the DNA within the tumorcells. The radiation is delivered in different ways. The most commoninvolves pointing a beam of radiation at the patient in a highly precisemanner, focusing on the tumor. To do this, a patient lies on a table andthe beam moves around him/her. The procedure lasts minutes, but may bedone daily for several weeks (depending on the type of tumor), toachieve a particular total prescribed dose.

Another radiation method sometimes employed, called brachytherapy,involves taking radioactive pellets (seeds) or wires and implanting themin the body in the area of the tumor. The implants can be temporary orpermanent. For permanent implants, the radiation in the seeds decaysover a period of days or weeks so that the patient is not radioactive.For temporary implants, the entire dose of radiation is usuallydelivered in a few days, and the patient must remain in the hospitalduring that time. For both types of brachytherapy, radiation isgenerally delivered to a very targeted area to gain local control over acancer (as opposed to treating the whole body, as chemotherapy does.)

Some highly selected patients may be referred for bone marrowtransplants. This procedure usually is performed either because apatient has a cancer that is particularly aggressive or because theyhave a cancer that has relapsed after being treated with conventionaltherapy. Bone marrow transplantation is a complicated procedure. Thereare many types, and they vary in their potential for causing sideeffects and cure. Most transplants are performed at special centers, andin many cases, their use is considered investigational.

A number of other therapies exist, although most of them are still beingexplored in clinical trials and have not yet become standard care.Examples include the use of immunotherapy, monoclonal antibodies,anti-angiogenesis factors and gene therapy.

Immunotherapy: There are various techniques designed to help thepatient's own immune system fight the cancer, quite separately fromradiation or chemotherapy. Oftentimes, to achieve the goal researchersinject the patient with a specially derived vaccine.

Monoclonal Antibodies: These are antibodies designed to attach tocancerous cells (and not normal cells) by taking advantage ofdifferences between cancerous and non-cancerous cells in their anitgenicand/or other. characteristics. The antibodies can be administered to thepatient alone or conjugated to various cytotoxic compounds or inradioactive form, such that the antibody preferentially targets thecancerous cells, thereby delivering the toxic agent or radioactivity tothe desired cells.

Anti-Angiogenesis Factors: As cancer cells rapidly divide and tumorsgrow, they can soon outgrow their blood supply. To compensate for this,some tumors secrete a substance believed to help induce the growth ofblood vessels in their vicinity, thus providing the cancer cells with avascular source of nutrients. Experimental therapies have been designedto arrest the growth of blood vessels to tumors.

Gene Therapy: Cancer is the product of a series of mutations thatultimately lead to the production of a cancer cell and its excessiveproliferation. Cancers can be treated by introducing genes to the cancercells that will act either to check or stop the cancer's proliferation,turn on the cell's programmed cell mechanisms to destroy the cell,enhance immune recognition of the cell, or express a pro-drug thatconverts to a toxic metabolite or a cytokine that inhibits tumor growth.

Benign tumors and malformations also can be treated by a variety ofmethods including surgery, radiotherapy, drug therapy, thermal orelectric ablation, cryotherapy, and others. Although benign tumors donot metastasize, they can grow large and they can recur. Surgicalextirpation of benign tumors has all the difficulties and side effectsof surgery in general and oftentimes must be repeatedly performed forsome benign tumors, such as for pituitary adenomas, meningeomas of thebrain, prostatic hyperplasia, and others.

Other conditions involving unwanted cellular elements exist whereselective cellular removal is desirable. For example, heart disease andstrokes commonly are caused by atherosclerosis, which is a proliferativelesion of fibrofatty and modified smooth muscle elements that distortthe blood vessel wall, narrow the lumen, constrict blood flow,predispose to focal blood clots, and ultimately lead to blockage andinfarction. There are various treatments for atherosclerosis such asbypass grafts; artificial grafts; angioplasty with recanalization,curettage, radiation, laser, or other removal; pharmacotherapy toinhibit atherosclerosis through lipid reduction; anti-clottingtherapies; and general measures of diet, exercise, and lifestyle. Amethod for removing atherosclerotic lesions without the risk and sideeffects of surgical procedures is needed.

Other examples of unwanted cellular elements where selective cellularremoval is desirable include viral induced growths, such as warts.Another example is hypertrophic inflammatory masses found ininflammatory conditions, and hypertrophic scars or keloids. Still otherexamples are found in cosmetic contexts such as the removal of unwantedhair, e.g., facial hair, or for shrinkage of unwanted tissue areas forcosmetic purposes, such as in the facial dermis and connective tissuesor in the dermas and connective tissue of the extremities.

Other examples of unwanted cellular elements where selective cellularremoval or the inhibition of cellular proliferation is desirable includestenosis and restenosis of any artery, valve or canal in the circulatorysystem including, but not limited to, valves (e.g., aortic stenosiswhich involves narrowing of the aortic valve orifice), coronary arteries(e.g., coronary ostial sclerosis which involves narrowing of the mouthsof the coronary arteries), carotid arteries, and renal arteries. Otherexamples include the inhibition or removal of unwanted cellular growthor accumulation causing the partial or complete occulsion of medicaldevices such as stents placed or implanted within a blood vessel fortreating stenoses, strictures or aneurysms therein or within the urinarytract and in bile ducts.

Still other examples will be obvious to those of ordinary skill in theart. In all or most of these examples there is a need for treatmentsthat can remove or destroy the unwanted cellular elements without therisks and side effects of conventional therapies or remove the unwantedcellular elements with more precision.

Throughout this description, including the foregoing description ofrelated art, any and all publicly available documents described herein,including any and all U.S. patents, are specifically incorporated byreference herein in their entirety. The foregoing description of relatedart is not intended in any way as an admission that any of the documentsdescribed therein, including pending U.S. patent applications, are priorart to the present disclosure. Moreover, the description herein of anydisadvantages associated with the described products, methods, and/orapparatus, is not intended to limit the embodiments. Indeed, aspects ofthe embodiments may include certain features of the described products,methods, and/or apparatus without suffering from their describeddisadvantages.

SUMMARY OF THE EMBODIMENTS

There remains a need in the art for new, less toxic treatments fortreating unwanted cellular elements. The embodiments satisfies theseneeds.

This disclosure is premised in part on the discovery that certainpeptides, including a specific peptide described by the amino acidsequenceIle-Asp-Gln-Gln-Val-Leu-Ser-Arg-Ile-Lys-Leu-Glu-Ile-Lys-Arg-Cys-Leu, arecapable of treating and/or killing unwanted cellular proliferations.These unwanted cellular proliferations include, inter alia, benign andmalignant tumors, glandular (e.g. prostate) hyperplasia, unwanted facialhair, warts, and unwanted fatty tissue.

The embodiments described herein are premised in part on the surprisingand unexpected discovery that certain peptide fragments and subsequencesof the peptideIle-Asp-Gln-Gln-Val-Leu-Ser-Arg-Ile-Lys-Leu-Glu-Ile-Lys-Arg-Cys-Leu(“SO5A Peptides”) also have the capability of treating and/or killingunwanted cellular proliferations.

Some embodiments are directed to methods of treating unwanted cellularproliferations (benign and malignant tumors, glandular (e.g. prostate)hyperplasia, unwanted facial hair, warts, and unwanted fatty tissue)comprising administering to a mammal in need thereof a therapeuticallyeffective amount of an S05A Peptide.

Such an S05A Peptide can be administered alone or conjugated to acarrier or an antibody. The S05A Peptides can be administeredintramuscularly, orally, intravenously, intraperitoneally,intracerebrally (intraparenchymally), intracerebroventricularly,intratumorally, intralesionally, intradermally, intrathecally,intranasally, intraocularly, intraarterially, topically, transdermally,via an aerosol, infusion, bolus injection, implantation device,sustained release system etc., either alone or conjugated to a carrier.Alternatively, the S05A Peptides can be expressed in vivo byadministering a gene that expresses the S05A Peptides, by administeringa vaccine that induces such production or by introducing cells, bacteriaor viruses that express the peptide in vivo, because of geneticmodification or otherwise.

In addition, the S05A Peptides may be used in conjunction with othertherapies for treating benign and malignant tumors and other unwanted orharmful cellular growths.

Both the foregoing general description and the following detaileddescription are exemplary and explanatory and are intended to providefurther explanation of the embodiments as claimed. Other objects,advantages, and features will be readily apparent to those skilled inthe art from the following detailed description of the embodiments.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Before the present proteins, nucleotide sequences, peptides, etc., andmethods are described, it is understood that this invention is notlimited to the particular methodology, protocols, cell lines, vectors,and reagents described, as these may vary. It also is to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to limit the scope ofthe present embodiments which will be limited only by the appendedclaims.

Terms and phrases used herein are defined as set forth below unlessotherwise specified.

Throughout this description, the singular forms “a,” “an,” and “the”include plural reference unless the context clearly dictates otherwise.Thus, for example, a reference to “a host cell” includes a plurality ofsuch host cells, and a reference to “an antibody” is a reference to oneor more antibodies and equivalents thereof known to those skilled in theart, and so forth.

Amino acids and amino acid residues described herein may be referred toaccording to the accepted one or three-letter code provided in the tablebelow.

TABLE 1 Three-Letter Amino One-Letter Acid Symbol Symbol Alanine A AlaArginine R Arg Asparagine N Asn Aspartic acid D Asp Cysteine C CysGlutamine Q Gln Glutamic acid E Glu Glycine G Gly Histidine H HisIsoleucine I Ile Leucine L Leu Lysine K Lys Methionine M MetPhenylalanine F Phe Proline P Pro Serine S Ser Threonine T ThrTryptophan W Trp Tyrosine Y Tyr Valine V Val

The term “peptide” as it is used herein, refers to a chain of at leasttwo amino acids and includes homologues, derivatives, fragments, andvariants of the peptide. The expression “S05A Peptide” refers to apeptide comprising at least one fragment or subsequence of the peptideSEQ ID NO. 1(Ile-Asp-Gln-Gln-Val-Leu-Ser-Arg-Ile-Lys-Leu-Glu-Ile-Lys-Arg-Cys-Leu)and includes any homologue, fragment, derivative, variant, fusionprotein, and peptide mimetics of the peptide unless the contextindicates otherwise. The expression “S05A Peptides” includes (but is notlimited to) peptides comprising at least one peptide selected from thegroup consisting of:

-   -   a) the peptide represented by the amino acid sequence in SEQ ID        NO. 2 (Ile-Asp-Gln-Gln-Val-Leu-Ser-Arg-Ile);    -   b) the peptide represented by the amino acid sequence in SEQ ID        NO. 3 (Lys-Leu-Glu-Ile-Lys-Arg-Cys-Leu);    -   c) the peptide represented by the amino acid sequence in SEQ ID        NO. 4 (Val-Leu-Ser-Arg-Ile-Lys);    -   d) the peptide represented by the amino acid sequence in SEQ ID        NO. 5 (Arg-Ile-Lys-Leu-Glu-Ile-Lys);    -   e) the peptide represented by the amino acid sequence in SEQ ID        NO. 6 (Val-Leu-Ser-Arg-Ile-Lys-Leu-Glu-Ile-Lys-Arg-Cys-Leu); and    -   f) the peptide represented by the amino acid sequence in SEQ ID        NO. 7 (Ile-Asp-Gln-Gln-Val-Leu-Ser-Arg-Ile-Lys-Leu-Glu-Ile).

The term “fragment” or “subsequence” refers to a protein or polypeptidethat consists of a continuous subsequence of the amino acid sequence ofa protein or peptide and includes naturally occurring fragments such assplice variants and fragments resulting from naturally occurring in vivoprotease activity. Such a fragment may be truncated at the aminoterminus, the carboxy terminus, and/or internally (such as by naturalsplicing). Such fragments may be prepared with or without an aminoterminal methionine. The term “fragment” includes fragments, whetheridentical or different, from the same protein or peptide, with acontiguous amino acid sequence in common or not, joined together, eitherdirectly or through a linker. As a consequence, any peptide thatincludes a fragment of SEQ ID NO. 1 can be any of those selected above,as well as other fragments or subsequences that, while not delineatedherein for purposes of brevity, will be readily apparent to thoseskilled in the art. The skilled artisan also will be capable ofselecting a suitable fragment for use in the embodiments without undueexperimentation using the guidelines and procedures outlined herein.

The term “variant” refers to a protein or polypeptide in which one ormore amino acid substitutions, deletions, and/or insertions are presentas compared to the amino acid sequence of an protein or peptide andincludes naturally occurring allelic variants or alternative splicevariants of an protein or peptide. The term “variant” includes thereplacement of one or more amino acids in a peptide sequence with asimilar or homologous amino acid(s) or a dissimilar amino acid(s). Thereare many scales on which amino acids can be ranked as similar orhomologous. (Gunnar von Heijne, Sequence Analysis in Molecular Biology,p. 123-39 (Academic Press, New York, N.Y. 1987.) Preferred variantsinclude alanine substitutions at one or more of amino acid positions.Other preferred substitutions include conservative substitutions thathave little or no effect on the overall net charge, polarity, orhydrophobicity of the protein. Conservative substitutions are set forthin Table 2 below.

TABLE 2 Conservative Amino Acid Substitutions Basic: arginine lysinehistidine Acidic: glutamic acid aspartic acid Uncharged Polar: glutamineasparagine serine threonine tyrosine Non-Polar: phenylalanine tryptophancysteine glycine alanine valine praline methionine leucine isoleucineTable 3 sets out another scheme of amino acid substitution:

TABLE 3 Original Residue Substitutions Ala gly; ser Arg lys Asn gln; hisAsp glu Cys ser Gln asn Glu asp Gly ala; pro His asn; gln Ile eu; valLeu ile; val Lys arg; gln; glu Met leu; tyr; ile Phe mat; leu; tyr Serthr Thr ser Trp tyr Tyr trp; phe Val ile; leu

Other variants can consist of less conservative amino acidsubstitutions, such as selecting residues that differ more significantlyin their effect on maintaining (a) the structure of the polypeptidebackbone in the area of the substitution, for example, as a sheet orhelical conformation, (b) the charge or hydrophobicity of the moleculeat the target site, or (c) the bulk of the side chain. The substitutionsthat in general are expected to have a more significant effect onfunction are those in which (a) glycine and/or proline is substituted byanother amino acid or is deleted or inserted; (b) a hydrophilic residue,e.g., seryl or threonyl, is substituted for (or by) a hydrophobicresidue, e.g., leucyl, isoleucyl, phenylalanyl, valyl, or alanyl; (c) acysteine residue is substituted for (or by) any other residue; (d) aresidue having an electropositive side chain, e.g., lysyl, arginyl, orhistidyl, is substituted for (or by) a residue having an electronegativecharge, e.g., glutamyl or aspartyl; or (e) a residue having a bulky sidechain, e.g., phenylalanine, is substituted for (or by) one not havingsuch a side chain, e.g., glycine. Other variants include those designedto either generate a novel glycosylation and/or phosphorylation site(s),or those designed to delete an existing glycosylation and/orphosphorylation site(s). Variants include at least one amino acidsubstitution at a glycosylation site, a proteolytic cleavage site and/ora cysteine residue. Variants also include proteins and peptides withadditional amino acid residues before or after the protein or peptideamino acid sequence on linker peptides. For example, a cysteine residuemay be added at both the amino and carboxy terminals of an S05A Peptidein order to allow the cyclisation of the Peptide by the formation of adi-sulphide bond. The term “variant” also encompasses polypeptides thathave the amino acid sequence of an S05A Peptide with at least one and upto 25 or more additional amino acids flanking either the 3′ or 5′ end ofthe Peptide.

The term “derivative” refers to a chemically modified protein orpolypeptide that has been chemically modified either by naturalprocesses, such as processing and other post-translationalmodifications, but also by chemical modification techniques, as forexample, by addition of one or more polyethylene glycol molecules,sugars, phosphates, and/or other such molecules, where the molecule ormolecules are not naturally attached to wild-type proteins or S05APeptides. Derivatives include salts. Such chemical modifications arewell described in basic texts and in more detailed monographs, as wellas in a voluminous research literature, and they are well known to thoseof skill in the art. It will be appreciated that the same type ofmodification may be present in the same or varying degree at severalsites in a given protein or polypeptide. Also, a given protein orpolypeptide may contain many types of modifications. Modifications canoccur anywhere in a protein or polypeptide, including the peptidebackbone, the amino acid side-chains, and the amino or carboxyl termini.Modifications include, for example, acetylation, acylation,ADP-ribosylation, amidation, covalent attachment of flavin, covalentattachment of a heme moiety, covalent attachment of a nucleotide ornucleotide derivative, covalent attachment of a lipid or lipidderivative, covalent attachment of phosphotidylinositol, cross-linking,cyclization, disulfide bond formation, demethylation, formation ofcovalent cross-links, formation of cysteine, formation of pyroglutamate,formylation, gamma-carboxylation, glycosylation, GPI anchor formation,hydroxylation, iodination, methylation, myristoylation, oxidation,proteolytic processing, phosphorylation, prenylation, racemization,glycosylation, lipid attachment, sulfation, gamma-carboxylation ofglutamic acid residues, hydroxylation and ADP-ribosylation,selenoylation, sulfation, transfer-RNA mediated addition of amino acidsto proteins, such as arginylation, and ubiquitination. See, forinstance, Proteins—Structure And Molecular Properties, 2nd Ed., T. E.Creighton, W. H. Freeman and Company, New York (1993) and Wold, F.,“Posttranslational Protein Modifications: Perspectives and Prospects,”pgs. 1-12 in Posttranslational Covalent Modification Of Proteins, B. C.Johnson, Ed., Academic Press, New York (1983); Seifter et al., Meth.Enzymol. 182:626-646 (1990) and Rattan et al., “Protein Synthesis:Posttranslational Modifications and Aging,” Ann. N.Y. Acad. Sci. 663:48-62 (1992). The term “derivatives” include chemical modificationsresulting in the protein or polypeptide becoming branched or cyclic,with or without branching. Cyclic, branched and branched circularproteins or polypeptides may result from post-translational naturalprocesses and may be made by entirely synthetic methods, as well.

The term “homologue” refers to a protein that is at least 60 percentidentical in its amino acid sequence of an S05A Peptide as determined bystandard methods that are commonly used to compare the similarity inposition of the amino acids of two polypeptides. The degree ofsimilarity or identity between two proteins can be readily calculated byknown methods, including but not limited to those described inComputational Molecular Biology, Lesk, A. M., ed., Oxford UniversityPress, New York; 1988; Biocomputing: Informatics and Genome Projects,Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis ofSequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., HumanaPress, New Jersey, 1994; Sequence Analysis in Molecular. Biology, vonHeinje, G., Academic Press, 1987; Sequence Analysis Primer, Gribskov, M.and Devereux, J., eds., M Stockton Press, New York, 1991; and Carillo H.and Lipman, D., SIAM, J. Applied Math., 48:1073 (1988). Preferredmethods to determine identity are designed to give the largest matchbetween the sequences tested. Methods to determine identity andsimilarity are codified in publicly available computer programs.

Preferred computer program methods useful in determining the identityand similarity between two sequences include, but are not limited to,the GCG program package (Devereux, J., et al., Nucleic Acids Research,12(1): 387 (1984)), BLASTP, BLASTN, and FASTA, Atschul, S. F. et al., J.Molec. Biol., 215: 403-410 (1990). The BLAST X program is publiclyavailable from NCBI and other sources (BLAST Manual, Altschul, S., etal., NCBI NLM NIH Bethesda, Md. 20894; Altschul, S., et al., J. Mol.Biol., 215: 403-410 (1990). By way of example, using a computeralgorithm such as GAP (Genetic Computer Group, University of Wisconsin,Madison, Wis.), the two proteins or polypeptides for which the percentsequence identity is to be determined are aligned for optimal matchingof their respective amino acids (the “matched span”, as determined bythe algorithm).

A gap opening penalty (which is calculated as 3 times the averagediagonal; the “average diagonal” is the average of the diagonal of thecomparison matrix being used; the “diagonal” is the score or numberassigned to each perfect amino acid match by the particular comparisonmatrix) and a gap extension penalty (which is usually 1/10 times the gapopening penalty), as well as a comparison matrix such as PAM 250 orBLOSUM 62 are used in conjunction with the algorithm. A standardcomparison matrix (see Dayhoff et al. in: Atlas of Protein Sequence andStructure, vol. 5, supp.3 for the PAM250 comparison matrix; see Henikoffet al., Proc. Natl. Acad. Sci USA, 89:10915-10919 for the BLOSUM 62comparison matrix) also may be used by the algorithm. The percentidentity then is calculated by the algorithm. Homologues will typicallyhave one or more amino acid substitutions, deletions, and/or insertionsas compared with the comparison protein or peptide, as the case may be.

The term “fusion protein” refers to a protein where one or more peptidesare recombinantly fused or chemically conjugated (including covalentlyand non-covalently) to a protein such as (but not limited to) anantibody or antibody fragment like an F.sub.ab fragment or short chainFv. The term “fusion protein” also refers to multimers (i.e. dimers,trimers, tetramers and higher multimers) of peptides. Such multimerscomprise homomeric multimers comprising one peptide, heteromericmultimers comprising more than one peptide, and heteromeric multimerscomprising at least one peptide and at least one other protein. Suchmultimers may be the result of hydrophobic, hyrdrophilic, ionic and/orcovalent associations, bonds or links, may be formed by cross-linksusing linker molecules or may be linked indirectly by, for example,liposome formation

The term “peptide mimetic” or “mimetic” refers to biologically activecompounds that mimic the biological activity of a peptide or a proteinbut are no longer peptidic in chemical nature, that is, they no longercontain any peptide bonds (that is, amide bonds between amino acids).Here, the term peptide mimetic is used in a broader sense to includemolecules that are no longer completely peptidic in nature, such aspseudo-peptides, semi-peptides and peptoids. Examples of peptidemimetics in this broader sense (where part of a peptide is replaced by astructure lacking peptide bonds) are described below. Whether completelyor partially non-peptide, peptide mimetics according to the embodimentsprovide a spatial arrangement of reactive chemical moieties that closelyresemble the three-dimensional arrangement of active groups in thepeptide on which the peptide mimetic is based. As a result of thissimilar active-site geometry, the peptide mimetic has effects onbiological systems that are similar to the biological activity of thepeptide.

The peptide mimetics of the embodiments are preferably substantiallysimilar in both three-dimensional shape and biological activity to thepeptides described herein. Examples of methods of structurally modifyinga peptide known in the art to create a peptide mimetic include theinversion of backbone chiral centers leading to D-amino acid residuestructures that may, particularly at the N-terminus, lead to enhancedstability for proteolytical degradation without adversely affectingactivity. An example is given in the paper “TritriatedD-ala.sup.1-Peptide T Binding”, Smith C. S. et al., Drug DevelopmentRes., 15, pp. 371-379 (1988). A second method is altering cyclicstructure for stability, such as N to C interchain imides and lactames(Ede et al. in Smith and Rivier (Eds.) “Peptides: Chemistry andBiology”, Escom, Leiden (1991), pp. 268-270). An example of this isgiven in conformationally restricted thymopentin-like compounds, such asthose disclosed in U.S. Pat. No. 4,457,489 (1985), Goldstein, G. et al.,the disclosure of which is incorporated by reference herein in itsentirety. A third method is to substitute peptide bonds in the peptideby pseudopeptide bonds that confer resistance to proteolysis.

A number of pseudopeptide bonds have been described that in general donot affect peptide structure and biological activity. One example ofthis approach is to substitute retro-inverso pseudopeptide bonds(“Biologically active retroinverso analogues of thymopentin”, Sisto A.et al in Rivier, J. E. and Marshall, G. R. (eds) “Peptides, Chemistry,Structure and Biology”, Escom, Leiden (1990), pp. 722-773) and Dalpozzo,et al. (1993), Int. J. Peptide Protein Res., 41:561-566, incorporatedherein by reference). According to this modification, the amino acidsequences of the peptides may be identical to the sequences of anpeptide described above, except that one or more of the peptide bondsare replaced by a retro-inverso pseudopeptide bond. Preferably the mostN-terminal peptide bond is substituted, since such a substitution willconfer resistance to proteolysis by exopeptidases acting on theN-terminus. Further modifications also can be made by replacing chemicalgroups of the amino acids with other chemical groups of similarstructure. Another suitable pseudopeptide bond that is known to enhancestability to enzymatic cleavage with no or little loss of biologicalactivity is the reduced isostere pseudopeptide bond (Couder, et al.(1993), Int. J. Peptide Protein Res., 41:181-184, incorporated herein byreference in its entirety).

Thus, the amino acid sequences of these peptides may be identical to thesequences of an peptide, except that one or more of the peptide bondsare replaced by an isostere pseudopeptide bond. Preferably the mostN-terminal peptide bond is substituted, since such a substitution wouldconfer resistance to proteolysis by exopeptidases acting on theN-terminus. The synthesis of peptides with one or more reduced isosterepseudopeptide bonds is known in the art (Couder, et al. (1993), citedabove). Other examples include the introduction of ketomethylene ormethylsulfide bonds to replace peptide bonds.

Peptoid derivatives of peptides represent another class of peptidemimetics that retain the important structural determinants forbiological activity, yet eliminate the peptide bonds, thereby conferringresistance to proteolysis (Simon, et al., 1992, Proc. Natl. Acad. Sci.USA, 89:9367-9371, incorporated herein by reference in its entirety).Peptoids are oligomers of N-substituted glycines. A number of N-alkylgroups have been described, each corresponding to the side chain of anatural amino acid (Simon, et al. (1992), cited above). Some or all ofthe amino acids of the peptides may be replaced with the N-substitutedglycine corresponding to the replaced amino acid.

The term “peptide mimetic” or “mimetic” also includes reverse-D peptidesand enantiomers as defined below.

The term “reverse-D peptide” refers to a biologically active protein orpeptide consisting of D-amino acids arranged in a reverse order ascompared to the L-amino acid sequence of an peptide. Thus, the carboxyterminal residue of an L-amino acid peptide becomes the amino terminalfor the D-amino acid peptide and so forth. For example, the peptide,ETESH, becomes H_(d)S_(d)E_(d)T_(d)E_(d), where E_(d), H_(d), S_(d), andT_(d) are the D-amino acids corresponding to the L-amino acids, E, H, S,and T respectively.

The term “enantiomer” refers to a biologically active protein or peptidewhere one or more the L-amino acid residues in the amino acid sequenceof an peptide is replaced with the corresponding D-amino acidresidue(s).

A “composition” as used herein, refers broadly to any compositioncontaining a recited peptide or amino acid sequence. The composition maycomprise a dry formulation, an aqueous solution, or a sterilecomposition. Compositions comprising peptides may be employed ashybridization probes. The probes may be stored in freeze-dried form andmay be associated with a stabilizing agent such as a carbohydrate. Inhybridizations, the probe may be deployed in an aqueous solutioncontaining salts, e.g., NaCl, detergents, e.g., sodium dodecyl sulfate(SDS), and other components, e.g., Denhardt's solution, dry milk, salmonsperm DNA, etc.

The embodiments are directed to a composition comprising S05A Peptidesas defined above in this embodiment.

Moreover, the embodiments includes other proteins that contain in wholeor part an S05A Peptide, whereby the proteins preferably possess thesame, similar, or enhanced bioactivity as the Peptide. Using theguidelines provided herein, a person ordinarily skilled in the art couldsynthesize specific proteins based on the amino acid sequence for anyS05A Peptide found to be an effective agent for causing cell death andtest them for efficacy as agents for causing cell death.

Other peptide sequences derived from an S05A Peptide found to be aneffective agent for causing cell death also may be effective agents forcausing cell death. A person ordinarily skilled in the art can, usingthe guidelines provided herein, synthesize without undue experimentationfragments of an effective Peptide spanning the entire amino acidsequence of that protein in order to identify other effective peptidesequences.

The S05A Peptides of the particularly preferred embodiments include, butare not limited to, the following:

SEQ ID NO. 2 IDQQVLSRI Ile-Asp-Gln-Gln-Val-Leu-Ser-Arg-Ile SEQ ID NO. 3KLEIKRCL Lys-Leu-Glu-Ile-Lys-Arg-Cys-Leu SEQ ID NO. 4 VLSRIKVal-Leu-Ser-Arg-Ile-Lys SEQ ID NO. 5 RIKLEIKArg-Ile-Lys-Leu-Glu-Ile-Lys-Arg SEQ ID NO. 6 VLSRIKLEIKRCLVal-Leu-Ser-Arg-Ile-Lys-Leu-Glu- Ile-Lys-Arg-Cys-Leu SEQ ID NO. 7IDQQVLSRIKLEI Ile-Asp-Gln-Gln-Val-Leu-Ser-Arg- Ile-Lys-Leu-Glu-Ile

It will be apparent to one of skill in the art that other smallerfragments of the above S05A Peptides may be selected such that thesepeptides will possess the same or similar biological activity. Otherfragments of may be selected by one skilled in the art such that thesepeptides will possess the same or similar biological activity. Thepeptides of the embodiments encompass these other fragments. In general,the peptides of the embodiments have at least 6 amino acids, preferablyat least 5 amino acids, and more preferably at least 4 amino acids.

The embodiments also encompasses S05A Peptides comprising two or moreS05A Peptides joined together. To the extent that an S05A Peptide hasthe desired biological activity, it follows that two such Peptides wouldalso possess the desired biological activity.

S05A Peptides and fragments, variants, derivatives, homologues, fusionproteins and mimetics thereof encompassed by this embodiment can beprepared using methods known to those of skill in the art, such asrecombinant DNA technology, protein synthesis and isolation of naturallyoccurring peptides, proteins, AD7c-protein and fragments, variants,derivatives and homologues thereof.

S05A Peptides and fragments, variants, derivatives, homologues, fusionproteins and mimetics thereof can be prepared from other peptides,proteins, and fragments, variants, derivatives and homologues thereofusing methods known to those having skill in the art. Such methodsinclude (but are not limited to) the use of proteases to cleave thepeptide, or protein into the desired S05A Peptides.

An S05A Peptide can be prepared using well known recombinant DNAtechnology methods such as those set forth in Sambrook et al. MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. and/or Ausubel et al., eds., Current Protocols inMolecular Biology, Green Publishers Inc. and Wiley and Sons, N.Y.

A gene or cDNA encoding an S05A Peptide may be obtained for example byscreening a genomic or cDNA library, or by PCR amplification. Probes orprimers useful for screening the library can be generated based onsequence information for other known genes or gene fragments from thesame or a related family of genes, such as, for example, conservedmotifs found in other peptides or proteins. In addition, where a geneencoding an S05A Peptide has been identified, all or a portion of thatgene may be used as a probe to identify homologous genes. The probes orprimers may be used to screen cDNA libraries from various tissue sourcesbelieved to express an S05A Peptide gene. Typically, conditions of highstringency will be employed for screening to minimize the number offalse positives obtained from the screen.

Another means to prepare a gene encoding an S05A Peptide is to employchemical synthesis using methods well known to the skilled artisan, suchas those described by Engels et al., Angew. Chem. Intl. Ed., 28:716-734.These methods include, inter alia, the phosphotriester, phosphoramidite,and H-phosphonate methods for nucleic acid synthesis. A preferred methodfor such chemical synthesis is polymer-supported synthesis usingstandard phosphoramidite chemistry. Typically, the DNA encoding anpeptide or protein will be several hundred nucleotides in length.Nucleic acids larger than about 100 to nucleotides can be synthesized asseveral fragments using these methods. The fragments then can be ligatedtogether to form the full length peptide or protein. Usually, the DNAfragment encoding the amino terminus of the protein will have an ATG,which encodes a methionine residue. This methionine may or may not bepresent on the mature form of the protein or peptide, depending onwhether the protein produced in the host cell is designed to be secretedfrom that cell.

The gene, cDNA, or fragment thereof encoding the S05A Peptide can beinserted into an appropriate expression or amplification vector usingstandard ligation techniques. The vector is typically selected to befunctional in the particular host cell employed (i.e., the vector iscompatible with the host cell machinery such that amplification of thegene and/or expression of the gene can occur). The gene, cDNA orfragment thereof encoding the S05A Peptide may be amplified/expressed inprokaryotic, yeast, insect (baculovirus systems) and/or eukaryotic hostcells. Selection of the host cell will depend in part on whether theS05A Peptide is to be glycosylated and/or phosphorylated. If so, yeast,insect, or mammalian host cells are preferable.

Typically, the vectors used in any of the host cells will contain atleast a 5′ flanking sequence (also referred to as a promoter) and otherregulatory elements as well, such as an enhancer(s), an origin ofreplication element, a transcriptional termination element, a completeintron sequence containing a donor and acceptor splice site, a signalpeptide sequence, a ribosome binding site element, a polyadenylationsequence, a polylinker region for inserting the nucleic acid encodingthe polypeptide to be expressed, and a selectable marker element. Eachof these elements is discussed below. Optionally, the vector may containa tag sequence, i.e., an oligonucleotide molecule located at the 5′ or3′ end of the protein or peptide coding sequence; the oligonucleotidemolecule encodes polyHis (such as hexaHis), or other tag such as FLAG,HA (hemaglutinin Influenza virus) or myc for which commerciallyavailable antibodies exist. This tag is typically fused to thepolypeptide upon expression of the polypeptide, and can serve as meansfor affinity purification of the protein or peptide from the host cell.Affinity purification can be accomplished, for example, by columnchromatography using antibodies against the tag as an affinity matrix.Optionally, the tag can subsequently be removed from the purifiedprotein or peptide by various means such as using certain peptidases.

The human immunoglobulin hinge and Fc region could be fused at eitherthe N-terminus or C-terminus of the S05A Peptide by one skilled in theart. The subsequent Fc-fusion protein could be purified by use of aProtein A affinity column. Fc is known to exhibit a long pharmacokinetichalf-life in vivo and proteins fused to Fc have been found to exhibit asubstantially greater half-life in vivo than the unfused counterpart.Also, fusion to the Fc region allows for dimerization/multimerization ofthe molecule that may be useful for the bioactivity of some molecules.

The 5′ flanking sequence may be homologous (i.e., from the same speciesand/or strain as the host cell), heterologous (i.e., from a speciesother than the host cell species or strain), hybrid (i.e., a combinationof 5′ flanking sequences from more than one source), synthetic, or itmay be the native protein or peptide gene 5′ flanking sequence. As such,the source of the 5′ flanking sequence may be any unicellularprokaryotic or eukaryotic organism, any vertebrate or invertebrateorganism, or any plant, provided that the 5′ flanking sequence isfunctional in, and can be activated by, the host cell machinery.

The 5′ flanking sequences useful in the vectors of this embodiment maybe obtained by any of several methods well known in the art. Typically,5′ flanking sequences useful herein other than the protein or peptidegene flanking sequence will have been previously identified by mappingand/or by restriction endonuclease digestion and can thus be isolatedfrom the proper tissue source using the appropriate restrictionendonucleases. In some cases, the full nucleotide sequence of the 5′flanking sequence may be known. Here, the 5′ flanking sequence may besynthesized using the methods described above for nucleic acid synthesisor cloning.

Where all or only a portion of the 5′ flanking sequence is known, it maybe obtained using PCR and/or by screening a genomic library withsuitable oligonucleotide and/or 5′ flanking sequence fragments from thesame or another species.

Where the 5′ flanking sequence is not known, a fragment of DNAcontaining a 5′ flanking sequence may be isolated from a larger piece ofDNA that may contain, for example, a coding sequence or even anothergene or genes. Isolation may be accomplished by restriction endonucleasedigestion using one or more carefully selected enzymes to isolate theproper DNA fragment. After digestion, the desired fragment may beisolated by agarose gel purification, Qiagen® column or other methodsknown to the skilled artisan. Selection of suitable enzymes toaccomplish this purpose will be readily apparent to one of ordinaryskill in the art.

The origin of replication element is typically a part of prokaryoticexpression vectors purchased commercially, and aids in the amplificationof the vector in a host cell. Amplification of the vector to a certaincopy number can, in some cases, be important for optimal expression ofthe protein or peptide. If the vector of choice does not contain anorigin of replication site, one may be chemically synthesized based on aknown sequence, and ligated into the vector. The transcriptiontermination element is typically located 3′ of the end of the protein orpeptide coding sequence and serves to terminate transcription of theprotein or peptide. Usually, the transcription termination element inprokaryotic cells is a G-C rich fragment followed by a poly T sequence.While the element may be cloned from a library or purchased commerciallyas part of a vector, it can also be readily synthesized using methodsfor nucleic acid synthesis such as those described above.

A selectable marker gene element encodes a protein necessary for thesurvival and growth of a host cell grown in a selective culture medium.Typical selection marker genes encode proteins that (a) conferresistance to antibiotics or other toxins, e.g., ampicillin,tetracycline, or kanamycin for prokaryotic host cells, (b) complementauxotrophic deficiencies of the cell; or (c) supply critical nutrientsnot available from complex media. Preferred selectable markers are thekanamycin resistance gene, the ampicillin resistance gene, and thetetracycline resistance gene.

The ribosome binding element, commonly called the Shine-Dalgarnosequence (prokaryotes) or the Kozak sequence (eukaryotes), is usuallynecessary for translation initiation of mRNA. The element is typicallylocated 3′ to the promoter and 5′ to the coding sequence of the proteinor peptide to be synthesized. The Shine-Dalgarno sequence is varied butis typically a polypurine (i.e., having a high A-G content). ManyShine-Dalgarno sequences have been identified, each of which can bereadily synthesized using methods set forth above and used in aprokaryotic vector.

In those cases where it is desirable for an S05A Peptide to be secretedfrom the host cell, a signal sequence may be used to direct the Peptideout of the host cell where it is synthesized, and the carboxy-terminalpart of the protein may be deleted in order to prevent membraneanchoring. Typically, the signal sequence is positioned in the codingregion of the S05A Peptide gene or cDNA, or directly at the 5′ end ofthe Peptide gene coding region. Many signal sequences have beenidentified, and any of them that are functional in the selected hostcell may be used in conjunction with the Peptide gene or cDNA.Therefore, the signal sequence may be homologous or heterologous to thePeptide gene or cDNA, and may be homologous or heterologous to thePeptide gene or cDNA. Additionally, the signal sequence may bechemically synthesized using methods set forth above. In most cases,secretion of the polypeptide from the host cell via the presence of asignal peptide will result in the removal of the amino terminalmethionine from the polypeptide.

In many cases, transcription of the S05A Peptide gene or cDNA isincreased by the presence of one or more introns in the vector; this isparticularly true where the Peptide is produced in eukaryotic hostcells, especially mammalian host cells. The introns used may benaturally occurring within the Peptide gene, especially where the geneused is a full length genomic sequence or a fragment thereof. Where theintron is not naturally occurring within the gene (as for most cDNAs),the intron(s) may be obtained from another source. The position of theintron with respect to the flanking sequence and the Peptide genegenerally is important, as the intron must be transcribed to beeffective. As such, where the Peptide gene inserted into the expressionvector is a cDNA molecule, the preferred position for the intron is 3′to the transcription start site, and 5′ to the polyA transcriptiontermination sequence. Preferably for Peptide cDNA, the intron or intronswill be located on one side or the other (i.e., 5′ or 3′) of the cDNAsuch that it does not interrupt this coding sequence. Any intron fromany source, including any viral, prokaryotic and eukaryotic (plant oranimal) organisms, may be used to practice this embodiment, providedthat it is compatible with the host cell(s) into which it is inserted.Also included herein are synthetic introns. Optionally, more than oneintron may be used in the vector.

Where one or more of the elements set forth above are not alreadypresent in the vector to be used, they may be individually obtained andligated into the vector. Methods used for obtaining each of the elementsare well known to the skilled artisan and are comparable to the methodsset forth above (i.e., synthesis of the DNA, library screening, and thelike).

The final vectors used to practice this embodiment may be constructedfrom starting vectors such as a commercially available vector. Suchvectors may or may not contain some of the elements to be included inthe completed vector. If none of the desired elements are present in thestarting vector, each element may be individually ligated into thevector by cutting the vector with the appropriate restrictionendonuclease(s) such that the ends of the element to be ligated in andthe ends of the vector are compatible for ligation. In some cases, itmay be necessary to blunt the ends to be ligated together in order toobtain a satisfactory ligation. Blunting is accomplished by firstfilling in “sticky ends” using Klenow DNA polymerase or T4 DNApolymerase in the presence of all four nucleotides. This procedure iswell known in the art and is described for example in Sambrook et al.,supra. Alternatively, two or more of the elements to be inserted intothe vector may first be ligated together (if they are to be positionedadjacent to each other) and then ligated into the vector.

An additional method for constructing the vector is to conduct allligations of the various elements simultaneously in one reactionmixture. Here, many nonsense or nonfunctional vectors will be generateddue to improper ligation or insertion of the elements, however thefunctional vector may be identified and selected by restrictionendonuclease digestion.

Preferred vectors for practicing this embodiment are those that arecompatible with bacterial, insect, and mammalian host cells. Suchvectors include, inter alia, pCRII, pCR3, and pcDNA3.1 (InvitrogenCompany, San Diego, Calif.), pBSII (Stratagene Company, La Jolla,Calif.), pET15b (Novagen, Madison, Wis.), PGEX (Pharmacia Biotech,Piscataway, N.J.), pEGFP-N2 (Clontech, Palo Alto, Calif.), pETL(BlueBachl; Invitrogen), and pFastBacDual (Gibco/BRL, Grand Island,N.Y.).

After the vector has been constructed and a nucleic acid moleculeencoding full length or truncated protein or peptide has been insertedinto the proper site of the vector, the completed vector may be insertedinto a suitable host cell for amplification and/or polypeptideexpression. Host cells may be prokaryotic host cells (such as E. coli)or eukaryotic host cells (such as a yeast cell, an insect cell, or avertebrate cell). The host cell, when cultured under appropriateconditions, can synthesize protein or peptide which can subsequently becollected from the culture medium (if the host cell secretes it into themedium) or directly from the host cell producing it (if it is notsecreted).

After collection, the S05A Peptide can be purified using methods such asmolecular sieve chromatography, affinity chromatography, and the like.Selection of the host cell for protein or peptide production will dependin part on whether the Peptide is to be glycosylated or phosphorylated(in which case eukaryotic host cells are preferred), and the manner inwhich the host cell is able to fold the Peptide into its native tertiarystructure (e.g., proper orientation of disulfide bridges, etc.) suchthat biologically active protein is prepared by the Peptide that hasbiological activity, the Peptide may be folded after synthesis usingappropriate chemical conditions as discussed below. Suitable cells orcell lines may be mammalian cells, such as Chinese hamster ovary cells(CHO), human embryonic kidney (HEK) 293, 293T cells, or 3T3 cells. Theselection of suitable mammalian host cells and methods fortransformation, culture, amplification, screening and product productionand purification are known in the art. Other suitable mammalian celllines, are the monkey COS-1 and COS-7 cell lines, and the CV-1 cellline. Further exemplary mammalian host cells include primate cell linesand rodent cell lines; including transformed cell lines. Normal diploidcells, cell strains derived from in vitro culture of primary tissue, aswell as primary explants, are also suitable. Candidate cells may begenotypically deficient in the selection gene, or may contain adominantly acting selection gene. Other suitable mammalian cell linesinclude but are not limited to, mouse neuroblastoma N2A cells, HeLa,mouse L-929 cells, 3T3 lines derived from Swiss, Balb-c or NIH mice, BHKor HaK hamster cell lines.

Similarly useful as host cells suitable for the present embodiments arebacterial cells. For example, the various strains of E. coli (e.g.,HB101, DH5.alpha., DH10, and MC1061) are well-known as host cells in thefield of biotechnology. Various strains of B. subtilis, Pseudomonasspp., other Bacillus spp., Streptomyces spp., and the like may also beemployed in this method. Many strains of yeast cells known to thoseskilled in the art also are available as host cells for expression ofthe polypeptides of the present embodiments.

Additionally, where desired, insect cell systems may be utilized in themethods of the present embodiments. Such systems are described forexample in Kitts et al. (Biotechniques, 14:810-817), Lucklow (Curr.Opin. Biotechnol., 4:564-572) and Lucklow et al. (J. Virol.,67:4566-4579). Preferred insect cells are Sf-9 and Hi5 (Invitrogen,Carlsbad, Calif.).

Insertion (also referred to as transformation or transfection) of thevector into the selected host cell may be accomplished using suchmethods as calcium chloride, electroporation, microinjection,lipofection, or the DEAE-dextran method. The method selected will inpart be a function of the type of host cell to be used. These methodsand other suitable methods are well known to the skilled artisan, andare set forth, for example, in Sambrook et al., supra.

The host cells containing the vector (i.e., transformed or transfected)may be cultured using standard media well known to the skilled artisan.The media will usually contain all nutrients necessary for the growthand survival of the cells. Suitable media for culturing E. coli cellsare for example, Luria Broth (LB) and/or Terrific Broth (TB). Suitablemedia for culturing eukaryotic cells are RPMI 1640, MEM, DMEM, all ofwhich may be supplemented with serum and/or growth factors as requiredby the particular cell line being cultured. A suitable medium for insectcultures is Grace's medium supplemented with yeastolate, lactalbuminhydrolysate, and/or fetal calf serum as necessary. Typically, anantibiotic or other compound useful for selective growth of thetransformed cells only is added as a supplement to the media. Thecompound to be used will be dictated by the selectable marker elementpresent on the plasmid with which the host cell was transformed. Forexample, where the selectable marker element is kanamycin resistance,the compound added to the culture medium will be kanamycin.

The amount of S05A Peptide produced in the host cell can be evaluatedusing standard methods known in the art. Such methods include, withoutlimitation, Western blot analysis, SDS-polyacrylamide gelelectrophoresis, non-denaturing gel electrophoresis, HPLC separation,mass spectroscopy, immunoprecipitation, and/or activity assays such asDNA binding gel shift assays.

If the protein or peptide has been designed to be secreted from the hostcells, the majority of the protein or peptide may be found in the cellculture medium. Proteins prepared in this way will typically not possessan amino terminal methionine, as it is removed during secretion from thecell. If however, the protein or peptide is not secreted from the hostcells, it will be present in the cytoplasm and/or the nucleus (foreukaryotic host cells) or in the cytosol (for gram negative bacteriahost cells) and may have an amino terminal methionine.

For an S05A Peptide situated in the host cell cytoplasm and/or nucleus,the host cells are typically first disrupted mechanically or withdetergent to release the intra-cellular contents into a bufferedsolution. The Peptide can then be isolated from this solution.

Purification of S05A Peptides from solution can be accomplished using avariety of techniques. If the S05A Peptide has been synthesized suchthat it contains a tag such as hexaHistidine (e.g. peptide/hexaHis) orother small peptide such as FLAG (Sigma-Aldritch, St. Louis, Mo.) orcalmodulin-binding peptide (Stratagene, La Jolla, Calif.) at either itscarboxyl or amino terminus, it may essentially be purified in a one-stepprocess by passing the solution through an affinity column where thecolumn matrix has a high affinity for the tag or for the proteindirectly (i.e., a monoclonal antibody specifically recognizing thepeptide). For example, polyhistidine binds with great affinity andspecificity to nickel, zinc and cobalt; thus immobilized metal ionaffinity chromatography which employs a nickel-based affinity resin (asused in Qiagen's QIAexpress system or Invitrogen's Xpress System) or acobalt-based affinity resin (as used in BD Biosciences-CLONTECH's Talonsystem) can be used for purification of peptide/polyHis. (See, forexample, Ausubel et al., eds., Current Protocols in Molecular Biology,Section 10.11.8, John Wiley & Sons, New York).

Where the S05A Peptide is prepared without a tag attached, and noantibodies are available, other well known procedures for purificationcan be used. Such procedures include, without limitation, ion exchangechromatography, hydroxyapatite chromatography, hydrophobic interactionchromatography, molecular sieve chromatography, HPLC, native gelelectrophoresis in combination with gel elution, and preparativeisoelectric focusing (Isoprime machine/technique, Hoefer Scientific). Insome cases, two or more of these techniques may be combined to achieveincreased purity.

If it is anticipated that the S05A Peptide will be found primarilyintracellularly, the intracellular material (including inclusion bodiesfor gram-negative bacteria) can be extracted from the host cell usingany standard technique known to the skilled artisan. For example, thehost cells can be lysed to release the contents of theperiplasm/cytoplasm by French press, homogenization, and/or sonicationfollowed by centrifugation. If the Peptide has formed inclusion bodiesin the cytosol, the inclusion bodies can often bind to the inner and/orouter cellular membranes and thus will be found primarily in the pelletmaterial after centrifugation. The pellet material then can be treatedat pH extremes or with chaotropic agent such as a detergent, guanidine,guanidine derivatives, urea, or urea derivatives in the presence of areducing agent such as dithiothreitol at alkaline pH or triscarboxyethyl phosphine at acid pH to release, break apart, andsolubilize the inclusion bodies. The Peptide in its now soluble form canthen be analyzed using gel electrophoresis, immunoprecipitation or thelike. If it is desired to isolate the Peptide, isolation may beaccomplished using standard methods such as those set forth below and inMarston et al. Meth. Enz., 182:264-275.

In some cases, the S05A Peptide may not be biologically active uponisolation. Various methods for refolding or converting the polypeptideto its tertiary structure and generating disulfide linkages, can be usedto restore biological activity. Such methods include exposing thesolubilized polypeptide to a pH usually above 7 and in the presence of aparticular concentration of a chaotrope. The selection of chaotrope isvery similar to the choices used for inclusion body solubilization butusually at a lower concentration and is not necessarily the samechaotrope as used for the solubilization. In most cases therefolding/oxidation solution will also contain a reducing agent or thereducing agent plus its, oxidized form in a specific ratio to generate aparticular redox potential allowing for disulfide shuffling to occur inthe formation of the protein's cysteine bridge(s). Some of the commonlyused redox couples include cysteine/cystamine, glutathione(GSH)/dithiobis GSH, cupric chloride, dithiothreitol(DTT)/dithiane DTT,2-mercaptoethanol(bME)/dithio-b(ME). In many instances a cosolvent isnecessary to increase the efficiency of the refolding and the morecommon reagents used for this purpose include glycerol, polyethyleneglycol of various molecular weights, and arginine.

If S05A Peptide inclusion bodies are not formed to a significant degreein the host cell, the S05A Peptide will be found primarily in thesupernatant after centrifugation of the cell homogenate, and the S05APeptide can be isolated from the supernatant using methods such as thoseset forth below.

In those situations where it is preferable to partially or completelyisolate the S05A Peptide, purification can be accomplished usingstandard methods well known to the skilled artisan. Such methodsinclude, without limitation, separation by electrophoresis followed byelectroelution, various types of chromatography (immunoaffinity,molecular sieve, and/or ion exchange), and/or high pressure liquidchromatography. In some cases, it may be preferable to use more than oneof these methods for complete purification.

In addition to preparing and purifying S05A Peptides using recombinantDNA techniques, the S05A Peptides and their fragments, variants,homologues, fusion proteins, peptide mimetics, and derivatives may beprepared by chemical synthesis methods (such as solid phase peptidesynthesis) using techniques known in the art such as those set forth byMerrifield et al., J. Am. Chem. Soc., 85:2149, Houghten et al. Proc NatlAcad. Sci. USA, 82:5132, and Stewart and Young, Solid Phase PeptideSynthesis, Pierce Chemical Co., Rockford, Ill. Such Peptides may besynthesized with or without a methionine on the amino terminus.Chemically synthesized S05A Peptides may be oxidized using methods setforth in these references to form disulfide bridges. The S05A Peptidesare expected to have biological activity comparable to Peptides producedrecombinantly or purified from natural sources, and thus may be used.interchangeably with recombinant or natural Peptide.

Chemically modified S05A Peptide compositions in which the Peptide islinked to a polymer are included within the scope of the presentembodiments. The polymer selected is typically water soluble so that theprotein to which it is attached does not precipitate in an aqueousenvironment, such as a physiological environment. The polymer selectedis usually modified to have a single reactive group, such as an activeester for acylation or an aldehyde for alkylation, so that the degree ofpolymerization may be controlled as provided for in the present methods.The polymer may be of any molecular weight, and may be branched orunbranched. Included within the scope of peptide polymers is a mixtureof polymers.

In some cases, it may be desirable to prepare nucleic acid and/or aminoacid variants of the naturally occurring S05A Peptides. Nucleic acidvariants may be produced using site directed mutagenesis, PCRamplification, or other appropriate methods, where the primer(s) havethe desired point mutations (see Sambrook et al., supra, and Ausubel etal., supra, for descriptions of mutagenesis techniques). Chemicalsynthesis using methods described by Engels et al., supra, may also beused to prepare such variants. Other methods known to the skilledartisan may be used as well.

Preferred nucleic acid variants are those containing nucleotidesubstitutions accounting for codon preference in the host cell that isto be used to produce S05A Peptides. Such codon optimization can bedetermined via computer algorithers which incorporate codon frequencytables such as Ecohigh. Cod for codon preference of highly expressedbacterial genes as provided by the University of Wisconsin PackageVersion 9.0, Genetics Computer Group, Madison, Wis. Other useful codonfrequency tables include Celegans_high.cod, Celegans_low.cod,Drosophila_high.cod, Human_high.cod, Maize_high.cod, and Yeast_high.cod.Other preferred variants are those encoding conservative amino acidchanges as described above (e.g., wherein the charge or polarity of thenaturally occurring amino acid side chain is not altered substantiallyby substitution with a different amino acid) as compared to wild type,and/or those designed to either generate a novel glycosylation and/orphosphorylation site(s), or those designed to delete an existingglycosylation and/or phosphorylation site(s).

S05A Peptides and fragments, homologs, variants, fusion proteins,peptide mimetics, derivatives and salts thereof also can be made usingconventional peptide synthesis techniques known to the skilled artisan.These techniques include chemical coupling methods (cf. Wunsch, E:“Methoden der organischen Chemie”, Volume 15, Band 1+2, Synthese vonPeptiden, thime Verlag, Stuttgart (1974), and Barrany, G.; Marrifield,R. B.: “The Peptides,” eds. E. Gross, J. Meienhofer, Volume 2, Chapter1, pp. 1-284, Academic Press (1980)), enzymatic coupling methods (cf.Widmer, F. Johansen, J. T., Carlsberg Res. Commun., Vol. 44, pp. 37-46(1979); Kullmann, W.: “Enzymatic Peptide Synthesis”, CRC Press Inc. BocaRaton, Fla. (1987); and Widmer, F., Johansen, J. T. in “SyntheticPeptides in Biology and Medicines,” eds. Alitalo, K., Partanen, P.,Vatieri, A., pp.79-86, Elsevier, Amsterdam (1985)), or a combination ofchemical and enzymatic methods if this is advantageous for the processdesign and economy. Using the guidelines provided herein, those skilledin the art are capable of varying the peptide sequence of the S05APeptide to make a homologue having the same or similar biologicalactivity (bioactivity) as the original or native S05A Peptide.

Advantages exist for using a mimetic of a given S05A Peptide rather thanthe Peptide itself. In general, peptide mimetics are more bioavailable,have a longer duration of action and can be cheaper to produce than thenative proteins and peptides.

Thus the peptides described above have utility in the development ofsuch small chemical compounds with similar biological activities andtherefore with similar therapeutic utilities. Peptide mimetics of S05APeptides can be developed using combinatorial chemistry techniques andother techniques known in the art (see e.g. Proceedings of the 20thEuropean Peptide Symposium, ed. G. Jung, E. Bayer, pp. 289-336, andreferences therein).

Examples of methods known in the art for structurally modifying apeptide to create a peptide mimetic include the inversion of backbonechiral centers leading to D-amino acid residue structures that may,particularly at the N-terminus, lead to enhanced stability forproteolytical degradation without adversely affecting activity. Anexample is provided in the paper “Tritriated D-ala.sup.1-Peptide TBinding”, Smith C. S. et al., Drug Development Res. 15, pp. 371-379(1988).

A second method is altering cyclic structure for stability, such as N toC interchain imides and lactames (Ede et al. in Smith and Rivier (Eds.)“Peptides: Chemistry and Biology”, Escom, Leiden (1991), pp. 268-270).An example of this is given in conformationally restrictedthymopentin-like compounds, such as those disclosed in U.S. Pat. No.4,457,489 (1985), Goldstein, G. et al., the disclosure of which isincorporated by reference herein in its entirety.

A third method is to substitute peptide bonds in the S05A Peptide bypseudopeptide bonds that confer resistance to proteolysis. A number ofpseudopeptide bonds have been described that in general do not affectpeptide structure and biological activity. One example of this approachis to substitute retro-inverso pseudopeptide bonds (“Biologically activeretroinverso analogues of thymopentin”, Sisto A. et al in Rivier, J. E.and Marshall, G. R. (eds) “Peptides, Chemistry, Structure and Biology”,Escom, Leiden (1990), pp. 722-773) and Dalpozzo, et al. (1993), Int. J.Peptide Protein Res., 41:561-566, incorporated herein by reference).According to this modification, the amino acid sequences of the peptidesmay be identical to the sequences of the peptides described above,except that one or more of the peptide bonds are replaced by aretro-inverso pseudopeptide bond. Preferably the most N-terminal peptidebond is substituted, since such a substitution will confer resistance toproteolysis by exopeptidases acting on the N-terminus.

The synthesis of peptides with one or more reduced retro-inversopseudopeptide bonds is known in the art (Sisto (1990) and Dalpozzo, etal. (1993), cited above). Thus, peptide bonds can be replaced bynon-peptide bonds that allow the peptide mimetic to adopt a similarstructure, and therefore biological activity, to the original peptide.Further modifications also can be made by replacing chemical groups ofthe amino acids with other chemical groups of similar structure. Anothersuitable pseudopeptide bond that is known to enhance stability toenzymatic cleavage with no or little loss of biological activity is thereduced isostere pseudopeptide bond is a (Couder, et al. (1993), Int. J.Peptide Protein Res., 41:181-184, incorporated herein by reference inits entirety). Thus, the amino acid sequences of these peptides may beidentical to the sequences of an peptide, except that one or more of thepeptide bonds are replaced by an isostere pseudopeptide bond. Preferablythe most N-terminal peptide bond is substituted, since such asubstitution would confer resistance to proteolysis by exopeptidasesacting on the N-terminus. The synthesis of peptides with one or morereduced isostere pseudopeptide bonds is known in the art (Couder, et al.(1993), cited above). Other examples include the introduction ofketomethylene or methylsulfide bonds to replace peptide bonds.

Peptoid derivatives of S05A Peptides represent another class of peptidemimetics that retain the important structural determinants forbiological activity, yet eliminate the peptide bonds, thereby conferringresistance to proteolysis (Simon, et al., 1992, Proc. Natl. Acad. Sci.USA, 89:9367-9371 and incorporated herein by reference in its entirety).Peptoids are oligomers of N-substituted glycines. A number of N-alkylgroups have been described, each corresponding to the side chain of anatural amino acid (Simon, et al. (1992), cited above and incorporatedherein by reference in its entirety). Some or all of the amino acids ofthe peptide are replaced with the N-substituted glycine corresponding tothe replaced amino acid.

The development of peptide mimetics can be aided by determining thetertiary structure of the original peptide by NMR spectroscopy,crystallography and/or computer-aided molecular modeling. Thesetechniques aid in the development of novel compositions of higherpotency and/or greater bioavailability and/or greater stability than theoriginal peptide (Dean (1994), BioEssays, 16: 683-687; Cohen andShatzmiller (1993), J. Mol. Graph., 11: 166-173; Wiley and Rich (1993),Med. Res. Rev., 13: 327-384; Moore (1994), Trends Pharmacol. Sci., 15:124-129; Hruby (1993), Biopolymers, 33: 1073-1082; Bugg et al. (1993),Sci. Am., 269: 92-98, all incorporated herein by reference in theirentirety).

Once a potential peptide mimetic compound is identified, it may besynthesized and assayed using the methods outlined in the examples belowto assess its activity. The peptide mimetic compounds obtained by theabove methods, having the biological activity of the peptides andsimilar three-dimensional structure, are encompassed by this embodiment.It will be readily apparent to one skilled in the art that a peptidemimetic can be generated from any of the peptides bearing one or more ofthe modifications described above. It will furthermore be apparent thatthe peptide mimetics of this embodiment can be further used for thedevelopment of even more potent non-peptidic compounds, in addition totheir utility as therapeutic compounds.

A number of organizations exist today that are capable of synthesizingthe peptides described herein. For example, given the sequence of anS05A Peptide, the organization can synthesize the Peptide and forwardthe synthesized Peptide with accompanying documentation and proof of theidentity of the Peptide.

Embodiments also encompasses the use of S05A Peptides and theircorresponding nucleic acid molecules for assays to test, eitherqualitatively or quantitatively, for the presence of S05A Peptides, S05APeptide DNA, or corresponding RNA in mammalian tissue or bodily fluidsamples. S05A Peptides and their corresponding nucleic acid moleculesmay have use in the preparation in such assays, whether or not thePeptide or the encoded Peptide DNA show biological activity. S05APeptide nucleic acid sequences may be a useful source of hybridizationprobes to test, either qualitatively or quantitatively, for the presenceof Peptide DNA or corresponding RNA in mammalian tissue or bodily fluidsamples. S05A Peptides which is not in itself biologically active may beuseful for preparing antibodies that recognize and/or bind to S05APeptides. Such antibodies may be prepared using standard methods. Thus,antibodies that react with or bind to the S05A Peptides, as well asshort chain antibody fragments and other reactive fragments of suchantibodies, also are contemplated as within the scope of the presentembodiments. The antibodies may be polyclonal, monoclonal, recombinant,chimeric, single-chain and/or bispecific. Typically, the antibody orfragment thereof will either be of human origin, or will be humanized,i.e., prepared so as to prevent or minimize an immune reaction to theantibody when administered to a patient. Preferred antibodies are humanantibodies, either polyclonal or monoclonal. The antibody fragment maybe any fragment that is reactive with peptides of the presentembodiments, such as, Fab, Fab′, etc. Also provided by this embodimentare the hybridomas generated by presenting any S05A Peptide as anantigen to a selected mammal, followed by fusing cells (e.g., spleencells) of the mammal with certain cancer cells to create immortalizedcell lines by known techniques. The methods employed to generate suchcell lines and antibodies directed against all or portions of an S05APeptide are also encompassed by the embodiments.

The antibodies may further be used for in vivo and in vitro diagnosticor research purposes, such as in labeled form to detect the presence ofan S05A Peptide in a body fluid or cell sample.

The embodiments also encompasses the use of one or more S05A Peptides ascalibration standards in assays that test, either qualitatively orquantitatively, for the presence of S05A Peptides, proteins, PeptideDNA, protein DNA, or corresponding RNA in mammalian tissue or bodilyfluid samples.

The present embodiments are directed to methods of treating conditionsrequiring removal of cells, such as benign and malignant tumors,glandular (e.g. prostate) hyperplasia, unwanted facial hair, warts, andunwanted fatty tissue, or the inhibition or prevention of unwantedcellular proliferation, such as stenosis of a stent. Such a methodcomprises administering to a mammal in need, or coating a device such asa stent with, a therapeutically effective amount of S05A Peptide.

The condition can be, for example, tumors of lung, breast, stomach,pancreas, prostate, bladder, bone, ovary, skin, kidney, sinus, colon,intestine, stomach, rectum, esophagus, blood, brain and its coverings,spinal cord and its coverings, muscle, connective tissue, adrenal,parathyroid, thyroid, uterus, testis, pituitary, reproductive organs,liver, gall bladder, eye, ear, nose, throat, tonsils, mouth, lymph nodesand lymphoid system, and other organs.

As used herein, the term “malignant tumor” is intended to encompass allforms of human carcinomas, sarcomas and melanomas which occur in thepoorly differentiated, moderately differentiated, andwell-differentiated forms.

This embodiment satisfies a need in the art for treatments that canremove benign tumors with less risk and fewer of the undesirable sideeffects of surgery. A method for removing benign tumors in surgicallyhazardous areas such as in deep locations in the body (e.g., brain,heart, lungs, and others) is particularly needed.

The method of treating conditions where cells must be removed can beused in conjunction with conventional methods of treating suchconditions, such as surgical excision, chemotherapy, and radiation.peptides can be administered before, during, or after such conventionaltreatments.

The condition to be treated can also be a hyperplasia, hypertrophy, orovergrowth of a tissue selected from the group consisting of lung,breast, stomach, pancreas, prostate, bladder, bone, ovary, skin, kidney,sinus, colon, intestine, stomach, rectum, esophagus, blood, brain andits coverings, spinal cord and its coverings, muscle, connective tissue,adrenal, parathyroid, thyroid, uterus, testis, pituitary, reproductiveorgans, liver, gall bladder, eye, ear, nose, throat, tonsils, mouth, andlymph nodes and lymphoid system.

Other conditions that can be treated using the method of the embodimentsare virally, bacterially, or parasitically altered tissue selected fromthe group consisting of lung, breast, stomach, pancreas, prostate,bladder, bone, ovary, skin, kidney, sinus, colon, intestine, stomach,rectum, esophagus, blood, brain and its coverings, spinal cord and itscoverings, muscle, connective tissue, adrenal, parathyroid, thyroid,uterus, testis, pituitary, reproductive organs, liver, gall bladder,eye, ear, nose, throat, tonsils, mouth, and lymph nodes and lymphoidsystem.

The condition to be treated can also be a malformation or disorder of atissue selected from the group consisting of lung, breast, stomach,pancreas, prostate, bladder, bone, ovary, skin, kidney, sinus, colon,intestine, stomach, rectum, esophagus, blood, brain and its coverings,spinal cord and its coverings, muscle, connective tissue, adrenal,parathyroid, thyroid, uterus, testis, pituitary, reproductive organs,liver, gall bladder, eye, ear, nose, throat, tonsils, mouth, and lymphnodes and lymphoid system.

In particular, the condition to be treated can be tonsillar hypertrophy,prostatic hyperplasia, psoriasis, eczema, dermatoses or hemorrhoids. Thecondition to be treated can be a vascular disease, such asatherosclerosis or arteriosclerosis, or a vascular disorder, such asvaricose veins, stenosis or restenosis of an artery or a stent. Thecondition to be treated can also be a cosmetic modification to a tissue,such as skin, eye, ear, nose, throat, mouth, muscle, connective tissue,hair, or breast tissue.

Therapeutic compositions of S05A Peptides also are contemplated in thepresent embodiments. Such compositions may comprise a therapeuticallyeffective amount of an S05A Peptide in admixture with a pharmaceuticallyacceptable carrier. The carrier material may be water for injection,preferably supplemented with other materials common in solutions foradministration to mammals. Typically, an S05A Peptide for therapeuticuse will be administered in the form of a composition comprisingpurified peptide in conjunction with one or more physiologicallyacceptable carriers, excipients, or diluents. Neutral buffered saline orsaline mixed with serum albumin are exemplary appropriate carriers.Preferably, the product is formulated as a lyophilizate usingappropriate excipients (e.g., sucrose). Other standard carriers,diluents, and excipients may be included as desired. Compositions of theembodiments also may comprise buffers known to those having ordinaryskill in the art with an appropriate range of pH values, including Trisbuffer of about pH 7.0-8.5, or acetate buffer of about pH 4.0-5.5, whichmay further include sorbitol or a suitable substitute therefor.

The use of S05A Peptides conjugated or linked or bound to an antibody,antibody fragment, antibody-like molecule, or a molecule with a highaffinity to a specific tumor marker, such as a cellular receptor, signalpeptide or over-expressed enzyme, for targeting to the unwanted cellularelements also is encompassed by the scope of the embodiments. Theantibody, antibody fragment, antibody-like molecule, or molecule with ahigh affinity to a specific tumor marker is used to target the Peptideconjugate to a specific cellular or tissue target. For example, a tumorwith a distinctive surface antigen or expressed antigen can be targetedby the antibody, antibody fragment, or antibody-like binding moleculeand the tumor cells can be killed by the Peptide. Such an approach usingantibody targeting has the anticipated advantages of decreasing dosage,increasing the likelihood of binding to and uptake by the target cells,and increased usefulness for targeting and treating metastatic tumorsand microscopic sized tumors.

This embodiment also encompasses the use of S05A Peptides conjugated orlinked or bound to a protein or other molecule to form a compositionthat, upon cleavage at or near the site(s) of the tumor or otherunwanted cells by a tumor- or site-specific enzyme or protease or by anantibody conjugate that targets tumor or other unwanted cells, releasesthe Peptide at or near the site(s) of the tumor or other unwanted cells

This embodiment also encompasses the use of S05A Peptides conjugated orlinked or bound to a protein or other molecule to form a compositionthat releases the Peptide or some biologically active fragment of thePeptide upon exposure of the tissue to be treated to light (as in lasertherapies or other photo-dynamic or photo-activated therapy), otherforms of electromagnetic radiation such as infra-red radiation,ultraviolet radiation, x-ray or gamma ray radiation, localized heat,alpha or beta radiation, ultrasonic emissions, or other sources oflocalized energy.

The S05A Peptides may be employed alone, together, or in combinationwith other pharmaceutical compositions, such as cytokines, growthfactors, antibiotics, apoptotis-inducing agents, anti-inflammatories,and/or chemotherapeutic agents as is appropriate for the indicationbeing treated.

This embodiment also encompasses therapeutic compositions of S05APeptides employing dendrimers, fullerenes, and other syntheticmolecules, polymers and macromolecules where the Peptide and/or itscorresponding DNA molecule is conjugated with, attached to or enclosedin the molecule, polymer or macromolecule, either by itself or inconjunction with other species of molecule such as a tumor-specificmarker. For example, U.S. Pat. No. 5,714,166, Bioactive and/or TargetedDendimer Conjugates, provides a method of preparing and using, interalia, dendritic polymer conjugates composed of at least one dendrimerwith a target director(s) and at least one bioactive agent conjugated toit. The disclosure of U.S. Pat. No. 5,714,166 is incorporated byreference herein in its entirety.

This embodiment also encompasses therapeutic compositions of S05APeptides and/or genes and drug delivery vehicles such as lipidemulsions, micelle polymers, polymer microspheres, electroactivepolymers, hydrogels and liposomes.

The use of S05A Peptides or related genes or gene equivalentstransferred to the unwanted cells also is encompassed by theembodiments. Overexpression of Peptide within the tumor can be used toinduce the cells in the tumor to die and thus reduce the tumor cellpopulation. The gene or gene equivalent transfer of S05A Peptide totreat the unwanted cellular elements is anticipated to have theadvantage of requiring less dosage, and of being passed on to thecellular progeny of the targeted cellular elements, thus necessitatingless frequent therapy, and less total therapy. This embodiment alsoencompasses the transfer of genes that code for a fusion proteincontaining an S05A Peptide to the unwanted cells or neighboring cellswhere, following the expression of the gene and the production and/orsecretion of the fusion protein, the fusion protein is cleaved either bynative enzymes or proteases or by a prodrug to release the Peptide in,at or near the unwanted cells.

The use of cloned recombinant peptide-antibody conjugates; clonedrecombinant peptide-antibody fragment conjugates; and cloned recombinantpeptide-antibody-like protein conjugates is also encompassed by thescope of the embodiments. The advantages of a cloned S05A Peptidecombined with targeting conjugate (such as an antibody, antibodyfragment, antibody-like molecule, or a molecule with a high affinity toa cancer-specific receptor or other tumor marker) are that such amolecule combines the targeting advantages described above in additionto advantages for manufacturing and standardized production of thecloned conjugated molecule.

This embodiment also encompasses the use of therapeutic compositions ofS05A Peptides or genes or gene equivalents as a component of the coatingof a medical device such as a stent in order to remove, inhibit orprevent unwanted cellular proliferation or accumulation.

Solid dosage forms for oral administration include but are not limitedto, capsules, tablets, pills, powders, and granules. In such soliddosage forms, the active compound is admixed with at least one of thefollowing: (a) one or more inert excipients (or carrier), such as sodiumcitrate or dicalcium phosphate; (b) fillers or extenders, such asstarches, lactose, sucrose, glucose, mannitol, and silicic acid; (c)binders, such as carboxymethylcellulose, alginates, gelatin,polyvinylpyrrolidone, sucrose and acacia; (d) humectants, such asglycerol; (e) disintegrating agents, such as agar-agar, calciumcarbonate, potato or tapioca starch, alginic acid, certain complexsilicates, and sodium carbonate; (f) solution retarders, such asparaffin; (g) absorption accelerators, such as quaternary ammoniumcompounds; (h) wetting agents, such as acetyl alcohol and glycerolmonostearate; (i) adsorbents, such as kaolin and bentonite; and (j)lubricants, such as talc, calcium stearate, magnesium stearate, solidpolyethylene glycols, sodium lauryl sulfate, or mixtures thereof. Forcapsules, tablets, and pills, the dosage forms may also comprisebuffering agents.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, solutions, suspensions, syrups, and elixirs. Inaddition to the active compounds, the liquid dosage forms may compriseinert diluents commonly used in the art, such as water or othersolvents, solubilizing agents, and emulsifiers. Exemplary emulsifiersare ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate,benzyl alcohol, benzyl benzoate, propyleneglycol, 1,3-butyleneglycol,dimethylformamide, oils, such as cottonseed oil, groundnut oil, corngerm oil, olive oil, castor oil, and sesame oil, glycerol,tetrahydrofurfuryl alcohol, polyethyleneglycols, fatty acid esters ofsorbitan, or mixtures of these substances, and the like.

Besides such inert diluents, the composition can also include adjuvants,such as wetting agents, emulsifying and suspending agents, sweetening,flavoring, and perfuming agents.

Actual dosage levels of active ingredients in the compositions of theembodiments may be varied to obtain an amount of S05A Peptide that iseffective to obtain a desired therapeutic response for a particularcomposition and method of administration. The selected dosage leveltherefore depends upon the desired therapeutic effect, the route ofadministration, the desired duration of treatment, and other factors.

With mammals, including humans, the effective amounts can beadministered on the basis of body surface area. The interrelationship ofdosages for animals of various sizes, species and humans (based onmg/M.sup.2 of body surface) is described by E. J. Freireich et al.,Cancer Chemother. Rep., 50 (4):219 (1966). Body surface area may beapproximately determined from the height and weight of an individual(see e.g., Scientific Tables, Geigy Pharmaceuticals, Ardsley, N.Y. pp.537-538 (1970)).

The total daily dose of the S05A Peptide administered to a host may bein single or divided doses. Dosage unit compositions may contain suchamounts of such submultiples thereof as may be used to make up the dailydose. It will be understood, however, that the specific dose level forany particular patient will depend upon a variety of factors includingthe body weight, general health, sex, diet, time and route ofadministration, potency of the administered drug, rates of absorptionand excretion, combination with other drugs and the severity of theparticular disease being treated.

A method of administering an S05A Peptide composition according to theembodiments includes, but is not limited to, administering the compoundsintramuscularly, orally, intravenously, intraperitoneally,intracerebrally (intraparenchymally), intracerebroventricularly,intratumorally, intralesionally, intradermally, intrathecally,intranasally, intraocularly, intraarterially, topically, transdermally,via an aerosol, infusion, bolus injection, implantation device,sustained release system etc.

Another method of administering an S05A Peptide of the embodiments is bya transdermal or transcutaneous route. One example of such an embodimentis the use of a patch. In particular, a patch can be prepared with afine suspension of Peptide in, for example, dimethylsulfoxide (DMSO), ora mixture of DMSO with cottonseed oil and brought into contact with theskin of the tumor carrying mammals away from the tumor location siteinside a skin pouch. Other mediums or mixtures thereof with othersolvents and solid supports would work equally as well. The patch cancontain the Peptide compound in the form of a solution or a suspension.The patch can then be applied to the skin of the patient, for example,by means of inserting it into a skin pouch of the patient formed byfolding and holding the skin together by means of stitches, clips orother holding devices. This pouch should be employed in such a manner sothat continuous contact with the skin is assured without theinterference of the mammal. Besides using a skin pouch, any device canbe used which ensures the firm placement of the patch in contact withthe skin. For instance, an adhesive bandage could be used to hold thepatch in place on the skin.

S05A Peptides may be administered in a sustained release formulation orpreparation. Suitable examples of sustained-release preparations includesemipermeable polymer matrices in the form of shaped articles, e.g.films, or microcapsules. Sustained release matrices include polyesters,hydrogels, polylactides (U.S. Pat. No. 3,773,919, EP 58,481), copolymersof L-glutamic acid and gamma ethyl-L-glutamate (Sidman et al.,Biopolymers, 22: 547-556), poly(2-hydroxyethyl-methacrylate) (Langer etal., J. Biomed. Mater. Res., 15: 167-277 and Langer, Chem. Tech., 12:98-105), ethylene vinyl acetate (Langer et al., supra) orpoly-D(-)-3-hydroxybutyric acid (EP 133,988). Sustained-releasecompositions also may include liposomes, which can be prepared by any ofseveral methods known in the art (e.g., Eppstein et al., Proc. Natl.Acad. Sci. USA, 82: 3688-3692; EP 36,676; EP 88,046; and EP 143,949).

Another method of administering an S05A Peptide of the embodiments is bydirect or indirect infusion of Peptide into the tumor or other tissue tobe treated. One example of such an embodiment is the direct injection ofPeptide into the tumor or other tissue to be treated. The treatment mayconsist of a single injection, multiple injections on one occasion or aseries of injections over a period of hours, days or months with theregression or destruction of the tumor or other tissue to be treatedbeing monitored by means of biopsy, imaging or other methods ofmonitoring tissue growth. The injection into the tumor or other tissueto be treated may be by a device inserted into an orifice such as thenose, mouth, ear, vagina, rectum or urethra or through an incision inorder to reach the tumor or tissue in vivo and may performed inconjunction with an imaging or optical system such as ultrasound orfibre optic scope in order to identify the appropriate site for theinjection(s). Another example of such an embodiment is the use of adevice that can provide a constant infusion of S05A Peptide to thetissue over time.

Another method of administering an S05A Peptide of the embodiments is inconjunction with a surgical or similar procedure employed to physicallyexcise, ablate or otherwise kill or destroy tumor or other tissue orcellular elements required or desired to be removed or destroyed whereinan S05A Peptide of the embodiments is administered to the immediatearea(s) surrounding the area(s) where the tumor or other tissue wasremoved in order to destroy or impede the growth of any tumor cells orother cellular elements not removed or destroyed by the procedure

Another method of administering an S05A Peptide of the embodiments is byimplantation of a device within the tumor or other tissue to be treated.One example of such an embodiment is the implantation of a wafercontaining Peptide in the tumor or other tissue to be treated. The waferreleases a therapeutic dose of Peptide into the tissue over time.Alternatively or additionally, the composition may be administeredlocally via implantation into the affected area of a membrane, sponge,or other appropriate material on to which the S05A Peptide has beenabsorbed. Where an implantation device is used, the device may beimplanted into any suitable tissue or organ, and delivery of the Peptidemay be directly through the device via bolus, or via continuousadministration, or via catheter using continuous infusion.

An alternative method of administration is to introduce one or morecopies of an S05A Peptide-encoding gene into the cell being targetedand, if necessary, inducing the copy(ies) of the gene to begin producingPeptide intracellularly. One manner in which gene therapy can be appliedis to use the S05A Peptide-encoding gene (either genomic DNA, cDNA,and/or synthetic DNA encoding the Peptide (or a fragment, variant,homologue or derivative thereof)) which may be operably linked to aconstitutive or inducible promoter to form a gene therapy DNA construct.The promoter may be homologous or heterologous to an endogenousPeptide-encoding gene, provided that it is active in the cell or tissuetype into which the construct will be inserted. Other components of thegene therapy DNA construct may optionally include, as required, DNAmolecules designed for site-specific integration (e.g., endogenousflanking sequences useful for homologous recombination), tissue-specificpromoter, enhancer(s) or silencer(s), DNA molecules capable of providinga selective advantage over the parent cell, DNA molecules useful aslabels to identify transformed cells, negative selection systems, cellspecific binding agents (as, for example, for cell targeting)cell-specific internalization factors, and transcription factors toenhance expression by a vector as well as factors to enable vectormanufacture.

Means of gene delivery to, a cell or tissue in vivo or ex vivo include(but are not limited to) direct injection of bare DNA, ballisticmethods, liposome-mediated transfer, receptor-mediated transfer(ligand-DNA complex), electroporation, and calcium phosphateprecipitation. See U.S. Pat. Nos. 4,970,154, WO 96/40958, U.S. Pat. No.5,679,559, U.S. Pat. No. 5,676,954, and U.S. Pat. No. 5,593,875, thedisclosures of each of which are incorporated by reference herein intheir entirety. They also include use of a viral vector such as aretrovirus, adenovirus, adeno-associated virus, pox virus, lentivirus,papilloma virus or herpes simplex virus, use of a DNA-protein conjugateand use of a liposome. The use of gene therapy vectors is described, forexample, in U.S. Pat. Nos. 5,672,344, U.S. Pat. No. 5,399,346, U.S. Pat.No.5,631,236, and U.S. Pat. No. 5,635,399, the disclosures of each ofwhich are incorporated by reference herein in their entirety.

The S05A Peptide-encoding gene may be delivered through implanting intopatients certain cells that have been genetically engineered ex vivo,using methods such as those described herein, to express and secrete theS05A Peptide or fragments, variants, homologues, or derivatives thereof.Such cells may be animal or human cells, and may be derived from thepatient's own tissue or from another source, either human or non-human.Optionally, the cells may be immortalized or be stem cells. However, inorder to decrease the chance of an immunological response, it ispreferred that the cells be encapsulated to avoid infiltration ofsurrounding tissues. The encapsulation materials are typicallybiocompatible, semi-permeable polymeric enclosures or membranes thatallow release of the protein product(s) but prevent destruction of thecells by the patient's immune system or by other detrimental factorsfrom the surrounding tissues. Methods used for membrane encapsulation ofcells are familiar to the skilled artisan, and preparation ofencapsulated cells and their implantation in patients may beaccomplished without undue experimentation. See, e.g., U.S. Pat. Nos.4,892,538; 5,011,472; and 5,106,627, the disclosures of each of whichare incorporated by reference herein in their entirety. A system forencapsulating living cells is described in PCT WO 91/10425. Techniquesfor formulating a variety of other sustained or controlled deliverymeans, such as liposome carriers, bio-erodible particles or beads, arealso known to those in the art, and are described, for example, in U.S.Pat. No. 5,653,975, the disclosure of which is incorporated by referenceherein in their entirety. The cells, with or without encapsulation, maybe implanted into suitable body tissues or organs of the patient.

Particularly preferred embodiments of methods of treating a disorderrequiring the removal or destruction of cells administering one or morepeptides of the embodiments, and the cells to be removed or destroyedare present lymphoid tissue. Other preferred embodiments includetreating conditions that require removal or destruction of cells such asany one selected from individually or in combination tonsillaryhypertrophy, prostatic hyperplasia, vascular disease (atherosclerosis orarteriosclerosis), hemorrhoids, varicose veins, psorasis, eczema,dermatosis, and a cosmetic modification to a tissue. Other treatableconditions include stenosis, restenosis, occulsion or blockage of anartery or of a stent placed or implanted in an artery. Suitable tissuethat can be treated in the preferred embodiments include skin, eye, ear,nose, throat, mouth, muscle, connective, hair, and breast.

Other preferred conditions treated in accordance with the embodimentsinclude those selected from an inflammatory disease, autoimmune disease,metabolic disease, hereditary/genetic disease, traumatic disease orphysical injury, nutritional deficiency disease, infectious disease,amyloid disease, fibrosis disease, storage disease, congenitalmalformation, enzyme deficiency disease, poisoning, intoxication,environmental disease, radiation disease, endocrine disease,degenerative disease and mechanical disease. In another preferredembodiment, the peptide is conjugated, linked, or bound to a moleculeselected from an antibody, antibody fragment, and an antibody-likebinding molecule, wherein said molecule has a higher affinity forbinding to a tumor or other target than binding to other cells. Inanother embodiment, the peptide is part of a single new clonedrecombinant molecule consisting of the peptide and a molecule selectedfrom the group consisting of an antibody, antibody fragment, andantibody-like binding molecule, wherein the molecule has a higheraffinity for binding to a tumor or other target than binding to othercells.

The following examples are provided to illustrate the presentembodiments. It should be understood, however, that the embodiments arenot to be limited to the specific conditions or details described inthese examples. Throughout the specification, any and all references toa publicly available document, including a U.S. patent, are specificallyincorporated by reference.

In particular, the embodiments expressly incorporate by reference theexamples contained in pending U.S. patent application Ser. No.10/092,934, Methods of Treating Tumors and Related Conditions UsingNeural Thread Proteins, which reveal that the whole AD7c-protein is aneffective agent for causing cell death both in vitro in glioma andneuroblastoma cell cultures and in vivo in normal rodent muscle tissue,subcutaneous connective tissue, and dermis and in a variety of differenthuman and non-human origin tumors, including mammary carcinoma, skincarcinoma and papilloma, colon carcinoma, glioma of brain, and others inrodent models. The embodiments also expressly incorporates by referencethe examples contained in pending U.S. patent applications Ser. No.10/153,334, entitled: Peptides Effective In The Treatment Of Tumors AndOther Conditions Requiring The Removal Or Destruction Of Cells; Ser. No.10/198,069, entitled: Peptides Effective In The Treatment Of Tumors AndOther Conditions Requiring The Removal Or Destruction Of Cells; Ser. No.10/198,070, entitled: Peptides Effective In The Treatment Of Tumors AndOther Conditions Requiring The Removal Or Destruction Of Cells, Ser. No.10/294, 891 entitled: Peptides Effective In The Treatment Of Tumors AndOther Conditions Requiring The Removal Or Destruction Of Cells; and Ser.No. 10/920,313 entitled: Peptides Effective In The Treatment Of TumorsAnd Other Conditions Requiring The Removal Or Destruction Of Cells, eachof which reveal that certain peptides specified therein are effectiveagents for causing cell death in vivo in normal rodent muscle tissue,subcutaneous connective tissue, dermis and other tissue.

EXAMPLE 1

The purpose of this example was to determine the effect of S05A Peptideson tissue at sites of injection.

The following S05A Peptides were synthesized using standard methods:

S05A-2 (SEQ ID NO. 2) IDQQVLSRI (Ile-Asp-Gln-Gln-Val-Leu-Ser-Arg-Ile);S05A-3 (SEQ ID NO. 3) KLEIKRCL (Lys-Leu-Glu-Ile-Lys-Arg-Cys-Leu); S05A-4(SEQ ID NO. 4) VLSRIK (Val-Leu-Ser-Arg-Ile-Lys); S05A-5 (SEQ ID NO. 5)RIKLEIK (Arg-Ile-Lys-Leu-Glu-Ile-Lys); S05A-6 (SEQ ID NO. 6)VLSRIKLEIKRCL (Val-Leu-Ser-Arg-Ile-Lys-Leu-Glu- Ile-Lys-Arg-Cys-Leu);S05A-7 (SEQ ID NO. 7) IDQQVLSRIKLEI (Ile-Asp-Gln-Gln-Val-Leu-Ser-Arg-Ile-Lys-Leu-Glu-Ile).

Male Sprague-Dawley rats (300 gram weight range) were anesthetized withether and given one of the above S05A Peptides by intraprostaticinfusion, a control injection of normal saline or no injection afteropen surgical visualization of the prostate. The injections consisted of300 μl of the S05A Peptide 1 mg/mL in PBS pH 7.4. (1.0 mg/kg) (n=36).Rats were painlessly sacrificed after 24 hours (n=12), 72 hours (n=12)and 7 days (n=12). Prostate glands were dissected, fixed in 10% bufferedformalin for 24 hours, embedded in paraffin, sectioned, and stained withH & E. For each animal the entire prostate gland was embedded andsectioned. All stained sections were examined histologically andmeasured. For each prostate at least 2 histological sections wereexamined, and for each histological section two cross-sectionaldiameters (D) at 90° from each other were measured (total of ≧8measurements per prostate). The mean of the cross-sectional diameters(D) for each group was used to estimate volume according to V=4/3π(D/2)³and the mean volume calculated for each group. Histological changes wereassessed on the following scale:

-   -   - Absent    -   + Present, Minimal    -   ++ Present, Moderate    -   +++ Present, Moderate and Diffuse    -   ++++ Present, Diffuse and Extensive

Results: Table 4 below sets out the histological changes of cell deathobserved.

TABLE 4 Histological Changes Histological Changes of Cell Death at 24 ofCell Death at 72 S05A Peptide Hours Hours S05A-2 ++++ ++++ S05A-3 + +S05A-4 + + S05A-5 + + S05A-6 + + S05A-7 ++++ ++++

Previous control studies of injections of 1 mg/mL PBS alone showedabsent or minimal histological changes, consisting of mild focalinflammation from the needles (see the examples contained in pendingU.S. patent application Ser. No.10/153,334, entitled: Peptides EffectiveIn The Treatment Of Tumors And Other Conditions Requiring The Removal OrDestruction Of Cells; Ser. No. 10/198,069, entitled: Peptides EffectiveIn The Treatment Of Tumors And Other Conditions Requiring The Removal OrDestruction Of Cells; Ser. No. 10/198,070, entitled: Peptides EffectiveIn The Treatment Of Tumors And Other Conditions Requiring The Removal OrDestruction Of Cells, Ser. No. 10/294, 891 entitled: Peptides EffectiveIn The Treatment Of Tumors And Other Conditions Requiring The Removal OrDestruction Of Cells; and Ser. No. 10/920,313 entitled: PeptidesEffective In The Treatment Of Tumors And Other Conditions Requiring TheRemoval Or Destruction Of Cells, incorporated by reference).

Table 5 below sets out the volume changes observed.

TABLE 5 S05A Calculated Mean Volume (mm³) Peptide 24 Hours 72 Hours 7Days Overall S05A-2 180 249 165 198 S05A-3 249 435 463 382 S05A-4 357408 333 366 S05A-5 249 408 392 350 S05A-6 310 493 408 404 S05A-7 180 165357 234

The overall reduction in prostate volume in S05A Peptides S05A-2 andS50A-7 injected rats was estimated to be on average >40% compared tocontrols. Rats treated with S50A-2 and S50A-7 showed extensive celldeath, necrosis, loss of glandular epithelium and atrophy. Controlsshowed minimal or absent changes consisting of acute inflammation at theinjection sites and focal microhemorrhages from the needles.

It will be apparent to those skilled in the art that variousmodifications and variations can be made in the methods and compositionsof the present embodiments without departing from the spirit or scope ofthe embodiments.

1. An isolated peptide consisting of a fragment of a second peptide,wherein the second peptide consists of the amino acid sequence in SEQ IDNO. 1(Ile-Asp-Gln-Gln-Val-Leu-Ser-Arg-Ile-Lys-Leu-Glu-Ile-Lys-Arg-Cys-Leu),wherein the fragment of the second peptide has at least 6 amino acids.2. A composition comprising at least one of the peptides as claimed inclaim 1 and a carrier.
 4. A mimetic of the peptide as claimed inclaim
 1. 5. An isolated peptide comprising as least two peptides ofclaim
 1. 6. An isolated peptide comprising at least two repetitions ofthe peptide of claim
 1. 8. An isolated peptide comprising the isolatedpeptide of claim 1 fused to an antibody, fragment of an antibody or anantibody-like molecule.
 9. An isolated peptide comprising the isolatedpeptide of claim 1 and at least one and up to 25 additional amino acidsflanking either the N-terminus or C-terminus of the isolated peptide ofclaim 1, wherein the isolated peptide does not comprise the peptideconsisting of the amino acid sequence in SEQ ID NO. 1(Ile-Asp-Gln-Gln-Val-Leu-Ser-Arg-Ile-Lys-Leu-Glu-Ile-Lys-Arg-Cys-Leu).10. A nucleic acid encoding an amino acid sequence corresponding to theisolated peptide of claim
 1. 11. A composition comprising one or morenucleic acid as claimed in claim 10 and a pharmaceutically acceptablecarrier.
 12. A method of treating a condition in a mammal requiringremoval or destruction of cells comprising administering to the mammal atherapeutically effective amount of the isolated peptide as claimed inclaim
 13. The method of claim 12, wherein the peptide is administered bya method selected from the group consisting of orally, subcutaneously,intradermally, intranasally, intravenously, intramuscularly,intrathecally, intranasally, intratumorally, topically, andtransdermally.
 14. The method of claim 12, wherein the method is carriedout on the mammal before, during, or after treatment of the mammal witha treatment selected from the group consisting of surgical excision,transplantation, grafting, chemotherapy, immunotherapy, vaccination,thermal or electrical ablation, cryotherapy, laser therapy,phototherapy, gene therapy, and radiation.
 15. The method of claim 12,wherein the condition is a benign or malignant tumor of a tissueselected from the group consisting of lung, breast, stomach, pancreas,prostate, bladder, bone, ovary, skin, kidney, sinus, colon, intestine,stomach, rectum, esophagus, heart, spleen, salivary gland, blood, brainand its coverings, spinal cord and its coverings, muscle, connectivetissue, adrenal, parathyroid, thyroid, uterus, testis, pituitary,reproductive organs, liver, gall bladder, eye, ear, nose, throat,tonsils, mouth, and lymph nodes and lymphoid system.
 16. The method ofclaim 12, wherein the condition is a hyperplasia, hypertrophy, orovergrowth of a tissue selected from the group consisting of lung,breast, stomach, pancreas, prostate, bladder, bone, ovary, skin, kidney,sinus, colon, intestine, stomach, rectum, esophagus, heart, spleen,salivary gland, blood, brain and its coverings, spinal cord and itscoverings, muscle, connective tissue, adrenal, parathyroid, thyroid,uterus, testis, pituitary, reproductive organs, liver, gall bladder,eye, ear, nose, throat, tonsils, mouth, and lymph nodes and lymphoidsystem.
 17. The method of claim 12, wherein the condition is a virally,bacterially, or parasitically altered tissue selected from the groupconsisting of lung, breast, stomach, pancreas, prostate, bladder, bone,ovary, skin, kidney, sinus, colon, intestine, stomach, rectum,esophagus, heart, spleen, salivary gland, blood, brain and itscoverings, spinal cord and its coverings, muscle, connective tissue,adrenal, parathyroid, thyroid, uterus, testis, pituitary, reproductiveorgans, liver, gall bladder, eye, ear, nose, throat, tonsils, mouth, andlymph nodes and lymphoid system.
 18. The method of claim 12, wherein thecondition is a malformation of a tissue selected from the groupconsisting of lung, breast, stomach, pancreas, prostate, bladder, bone,ovary, skin, kidney, sinus, colon, intestine, stomach, rectum,esophagus, heart, spleen, salivary gland, blood, brain and itscoverings, spinal cord and its coverings, muscle, connective tissue,adrenal, parathyroid, thyroid, uterus, testis, pituitary, reproductiveorgans, liver, gall bladder, eye, ear, nose, throat, tonsils, mouth, andlymph nodes and lymphoid system.
 19. A method of preventing orinhibiting the stenosis, occulsion or blockage of a stent comprisingcoating the stent with at least a therapeutically effective amount ofthe isolated peptide as claimed in claim 1.